Articular cartilage injury represents one of the leading causes of disability. Current treatments for repairing cartilage defects rely mostly on surgical intervention to restore articular surface, yet often result in undesirable fibrocartilage formation with poor long-term outcomes. While both synthetic and natural hydrogels have shown efficacy in supporting MSC chondrogenesis in 3D, conventional hydrogels generally lead to slow neocartilage deposition by MSCs in 3D restricted to peri-cellular regions, resulting in long delay before significant increase in mechanical strength. Given articular cartilage is a weight-bearing tissue, there remains a critical need for novel scaffolds to accelerate cartilage deposition by MSCs in 3D with improved mechanical strength. During cartilage development, cell-matrix and cell-cell adhesions play an important role in the mesenchymal condensation process, which involves multiple ligand types and cell-mediated ligand clustering. However, conventional covalently crosslinked hydrogels are characterized by fixed crosslinks and ligands, which do not allow cells to re-organize the surrounding niche cues. To address the above limitations, our group has recently reported sliding hydrogels with mobile crosslinks and biochemical ligands as a 3D niche. The molecular mobility of the sliding hydrogels enables the cells to reorganize ligands and cytoskeleton in 3D, and substantially accelerates cartilage matrix production with improved mechanical strength than conventional non- mobile hydrogels. We hypothesize that: (1) increasing molecular mobility will accelerate chondrogenesis and neocartilage deposition by human MSCs in 3D; and (2) varying the type and density of ligands will further enhance cartilage formation by MSCs with improved mechanical strength in vitro and in vivo. By fusing principles and tools from material science, stem cell biology, animal models and imaging, we have assembled a multidisciplinary team of basic and clinician scientists to test the above hypotheses by pursuing the following aims: Aim 1: Develop and characterize sliding hydrogels with tunable molecular mobility and biochemical ligands as a 3D stem-cell niche. Aim 2. Examine the impact of varying molecular mobility on modulating the speed and quality of neocartilage formation by MSCs in 3D sliding hydrogels. Aim 3. Evaluate the effect of varying the types and density of mobile biochemical ligands on MSC-based cartilage tissue formation, and explore potential molecular mechanisms by which molecular mobility modulate stem cell fates in 3D. Aim 4: Assess the role of the molecular mobility of sliding hydrogels in accelerating MSC-based cartilage formation in vivo using a rat osteochondral defect model. Upon completion of the project, we expect to identify sliding hydrogels with optimized molecular mobility and biochemical ligands as novel matrices to accelerate the speed of cartilage regeneration by MSCs with substantially improved mechanical strength. The outcomes of the proposed aims will establish molecular mobility as a novel parameter in 3D niche design, and shed lights on potential molecular mechanisms that lead to molecular mobility-enhanced cartilage regeneration.